Abstract

Unwanted sedimentation and attachment of a number of cells onto the bottom channel often occur on relatively large-scale inlets of conventional microfluidic channels as a result of gravity and fluid shear. Phenomena such as sedimentation have become recognized problems that can be overcome by performing microfluidic experiments properly, such as by calculating a meaningful output efficiency with respect to real input. Here, we present a dual-inlet design method for reducing cell loss at the inlet of channels by adding a new “ upstream inlet ” to a single main inlet design. The simple addition of an upstream inlet can create a vertically layered sheath flow prior to the main inlet for cell loading. The bottom layer flow plays a critical role in preventing the cells from attaching to the bottom of the channel entrance, resulting in a low possibility of cellsedimentation at the main channel entrance. To provide proof-of-concept validation, we applied our design to a microfabricated flow cytometer system (μFCS) and compared the cell counting efficiency of the proposed μFCS with that of the previous single-inlet μFCS and conventional FCS. We used human white blood cells and fluorescent microspheres to quantitatively evaluate the rate of cellsedimentation in the main inlet and to measure fluorescence sensitivity at the detection zone of the flow cytometer microchip. Generating a sheath flow as the bottom layer was meaningfully used to reduce the depth of field as well as the relative deviation of targets in the z-direction (compared to the x-yflow plane), leading to an increased counting sensitivity of fluorescent detection signals. Counting results using fluorescent microspheres showed both a 40% reduction in the rate of sedimentation and a 2-fold higher sensitivity in comparison with the single-inlet μFCS. The results of CD4+ T-cell counting also showed that the proposed design results in a 25% decrease in the rate of cellsedimentation and a 28% increase in sensitivity when compared to the single-inlet μFCS. This method is simple and easy to use in design, yet requires no additional time or cost in fabrication. Furthermore, we expect that this approach could potentially be helpful for calculating exact cell loading and counting efficiency for a small input number of cells, such as primary cells and rare cells, in microfluidic channel applications.

This work was supported by a grant from the Kyung Hee University in 2013 (KHU-20130431). This research was also supported by the National Research Foundation of Korea (NRF 2014023680) and Basic Science Research Program through the NRF, funded by the Ministry of Science, ICT and Future Planning (2014001331).

Article outline:INTRODUCTIONMATERIALS AND METHODSSetup of the flow cytometry system and its peripheralsPreparation of a microfabricated flow cytometer microchipPreparation of human white blood cells for flow cytometric testRESULTSThe concept of dual inlet is firmly supported by fluid force theoriesThe dual inlet generates a sheath flow as the bottom layer, leading to a low possibility of cellsedimentationThe dual inlet increases both cell loading and counting efficiencyDISCUSSIONThe simple dual inlet can overcome a known dilemma in microchannelsThe cell loading efficiency can be calibrated with actual cell number with the aid of a dual inletImplication of using the dual inlet for cell analysis in microfluidicsOther applications of the use of dual inlet in microfluidicsLimitations of the proposed method and systemCONCLUSIONS